Protein kinase Hsl1 phosphorylates Pah1 to inhibit phosphatidate phosphatase activity and regulate lipid synthesis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, plays a key role in utilizing PA for the synthesis of the neutral lipid triacylglycerol and thereby controlling the PA-derived membrane phospholipids. The enzyme function is controlled by its subcellular location as regulated by phosphorylation and dephosphorylation. Pah1 is initially inactivated in the cytosol through phosphorylation by multiple protein kinases and then activated via its recruitment and dephosphorylation by the protein phosphatase Nem1-Spo7 at the nuclear/endoplasmic reticulum membrane where the PA phosphatase reaction occurs. Many of the protein kinases that phosphorylate Pah1 have yet to be characterized with the identification of the target residues. Here, we established Pah1 as a bona fide substrate of septin-associated Hsl1, a protein kinase involved in mitotic morphogenesis checkpoint signaling. The Hsl1 activity on Pah1 was dependent on reaction time and the amounts of protein kinase, Pah1, and ATP. The Hsl1 phosphorylation of Pah1 occurred on Ser-748 and Ser-773, and the phosphorylated protein exhibited a 5-fold reduction in PA phosphatase catalytic efficiency. Analysis of cells expressing the S748A and S773A mutant forms of Pah1 indicated that Hsl1-mediated phosphorylation of Pah1 promotes membrane phospholipid synthesis at the expense of triacylglycerol, and ensures the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase. This work advances the understanding of how Hsl1 facilitates membrane phospholipid synthesis through the phosphorylation-mediated regulation of Pah1.

In Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase, which catalyzes the Mg 2+ -dependent dephosphorylation of PA to produce diacylglycerol, plays a key role in utilizing PA for the synthesis of the neutral lipid triacylglycerol and thereby controlling the PA-derived membrane phospholipids.The enzyme function is controlled by its subcellular location as regulated by phosphorylation and dephosphorylation.Pah1 is initially inactivated in the cytosol through phosphorylation by multiple protein kinases and then activated via its recruitment and dephosphorylation by the protein phosphatase Nem1-Spo7 at the nuclear/endoplasmic reticulum membrane where the PA phosphatase reaction occurs.Many of the protein kinases that phosphorylate Pah1 have yet to be characterized with the identification of the target residues.Here, we established Pah1 as a bona fide substrate of septinassociated Hsl1, a protein kinase involved in mitotic morphogenesis checkpoint signaling.The Hsl1 activity on Pah1 was dependent on reaction time and the amounts of protein kinase, Pah1, and ATP.The Hsl1 phosphorylation of Pah1 occurred on Ser-748 and Ser-773, and the phosphorylated protein exhibited a 5-fold reduction in PA phosphatase catalytic efficiency.Analysis of cells expressing the S748A and S773A mutant forms of Pah1 indicated that Hsl1-mediated phosphorylation of Pah1 promotes membrane phospholipid synthesis at the expense of triacylglycerol, and ensures the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase.This work advances the understanding of how Hsl1 facilitates membrane phospholipid synthesis through the phosphorylation-mediated regulation of Pah1.
Pah1 is a peripheral membrane enzyme and its PAP function occurs at the nuclear/endoplasmic reticulum (ER) membrane surface (30,47,48).The subcellular localization of Pah1 is controlled by the posttranslational modifications of phosphorylation and dephosphorylation (49).Pah1 is phosphorylated by multiple protein kinases (50-56) (Fig 2A ), and the phosphorylated enzyme is localized to the cytosol (50,57).
In contrast to Pah1 phosphorylation, its dephosphorylation is catalyzed by a single protein phosphatase complex that is composed of the Nem1 (catalytic) and Spo7 (regulatory) subunits (34,57,60,61) (Fig. 2B).Nem1-Spo7 has the function of activating Pah1; it recruits Pah1 to the nuclear/ER membrane and dephosphorylates the enzyme (34,48,49,57,61,62).The dephosphorylation permits Pah1 to hop onto and scoot along the membrane to recognize its substrate PA and catalyze the PAP reaction (63).In a feedforward mechanism, PA stimulates Nem1-Spo7 phosphatase activity (64).Additionally, the dephosphorylation of Pah1 stimulates its PAP activity (61).Like Pah1, the Nem1 and Spo7 subunits are subject to phosphorylation by protein kinases A and C (65, 66) (Fig. 2B).The phosphorylations of the phosphatase complex by these protein kinases have opposing effects on Pah1 activity and its cellular function such as TAG synthesis; protein kinase A inhibits the protein phosphatase (65), whereas protein kinase C stimulates the enzyme (66).Consistent with the requirement of the phosphatase complex for Pah1 localization and PAP activity, the nem1D or spo7D mutation elicits the same phenotypes characteristic of those caused by the pah1D mutation (60,67,68).
Phosphoproteomics studies have shown that Pah1 is phosphorylated at 56 sites (Fig. 2A) and a substrate for 20 protein kinases (49,69).The protein kinases that have been

Dgk1
Lipid droplets The DAG produced by the PAP reaction may be used for the synthesis of phosphatidylcholine and phosphatidylethanolamine via the CDP-choline and CDPethanolamine branches, respectively, of the Kennedy pathway when cells are supplemented with choline and/or ethanolamine.In addition to its role in lipid synthesis, PA signals the transcriptional regulation of phospholipid synthesis genes via the Henry regulatory circuit.More comprehensive pathways of lipid synthesis, along with details of the Henry regulatory circuit may be found in Refs.(5,6).CL, cardiolipin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine.

Kennedy pathway
Hsl1 phosphorylates PA phosphatase Pah1 characterized for Pah1 phosphorylation with the identification of target sites include the cyclin-dependent kinases Cdc28 and Pho85, casein kinases I and II, protein kinases A and C, and the glycogen synthase kinase 3b homolog Rim11 (50-56) (Fig. 2A).This information along with analyses of cells bearing Pah1 deficient in phosphorylation at the kinase-specific sites has provided insight into the role of each protein kinase in the regulation of Pah1 localization, catalytic activity, and stability (49).Many of the protein kinases that phosphorylate Pah1 have yet to be characterized and their target sites of phosphorylation need to be defined.In this study, we focused on the septin-associated protein kinase Hsl1 (histone synthetic le-thal1), one of the kinases shown to phosphorylate Pah1 in the global analysis of yeast protein phosphorylation (69).Hsl1 is involved in the S. cerevisiae morphogenesis checkpoint, a mechanism that ensures the presence of a bud prior to progression through the mitotic phase of the cell cycle (70)(71)(72).As the morphogenesis checkpoint function is relevant to the exponential growth during which Pah1 is highly phosphorylated (49), we hypothesized that the Hsl1 phosphorylation of Pah1 would have an inhibitory effect on PAP activity and promote the synthesis of membrane phospholipids.Here, we established that Pah1 is a bona fide substrate of Hsl1 and that its phosphorylation on Ser-748 and Ser-773 has an inhibitory effect on PAP activity.Mutational analysis of Ser-748 and Ser-773 indicated that the Hsl1-mediated phosphorylation at these sites facilitates membrane phospholipid synthesis at the expense of triacylglycerol, and ensures the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase.

Growth-dependent expression and purification of Hsl1
Hsl1 fused with a TAP tag at the C-terminus was isolated from the extracts of early exponential-phase cells by affinity chromatography with IgG-Sepharose.Since the TAP tag was resistant to cleavage by TEV protease, the protein kinase was purified without removing the affinity tag.Hsl1 was expressed at a low level and easily degraded, and the yield of its purification was 1 mg per ml (Fig 3A).The identity of Hsl1-TAP migrating at the estimated molecular mass of 190 kDa was confirmed by immunoblotting with an antibody against calmodulin-binding peptide of the affinity tag (73, 74) (Fig. 3A) as well as by LC-MS/MS analysis of the fusion protein-derived peptides (61% coverage of the protein) (Table S1).
The effect of growth on the level of Hsl1 was examined to better understand the role of the protein kinase in the regulation of Pah1.The S. cerevisiae cells expressing the TAPtagged chromosomal HSL1 were grown in an SC medium and analyzed for the level of Hsl1-TAP by immunoblotting with an anti-calmodulin-binding peptide antibody.During cell growth, the cellular level of the protein kinase was highest at the early exponential phase (A 600 = 0.08) and decreased to the undetectable at the late exponential phase (A 600 = 1.32) (Fig. 3B).

Hsl1 phosphorylates Pah1 on Ser-748 and Ser-773
To examine the phosphorylation of Pah1 by the purified Hsl1, we utilized the His-tagged Pah1 purified from heterologous expression in Escherichia coli (30).The Hsl1 protein A, the schematic shows the domains/regions of Pah1.The N-LIP and the haloacid dehalogenase (HAD)-like domains are required for PAP activity (30,41).The N-terminal amphipathic helix (AH) is responsible for interaction with the membrane (48).The regulation of the phosphorylation (RP) domain facilitates phosphorylation (93).The essential tryptophan contained within the WRDPLVDID domain, which is C-terminal to the HAD-like domain (138), is required for the in vivo catalytic function (89,138).The acidic tail (AT) associates with the Nem1-Spo7 complex (62) through ionic interaction with the Cterminal basic tail of Spo7 (139).The intrinsically disordered regions (IDRs) contain almost all of the sites of phosphorylation that serve for interaction with the Nem1-Spo7 complex.The serine (S) and threonine (T) residues known to be phosphorylated (50-57, 75, 83, 84, 89, 140-147) are grouped at their approximate regions in the Pah1 protein.The sites phosphorylated by casein kinase I (CKI) (55), casein kinase II (CKII) (54), Cdc28 (51), Hsl1 (this study), Pho85 (50), protein kinase A (PKA) (52), protein kinase C (PKC) (53), and Rim11 (56) are indicated.B, the positive (solid green arrow) or negative (solid blunted red line) impacts of Pah1 phosphorylation (denoted by the small black circles) by the indicated protein kinases and Pah1 dephosphorylation by Nem1-Spo7, whose components are also subject to phosphorylation by protein kinases A and C (65, 66), are indicated.The casein kinase I phosphorylation of Pah1 stimulates its subsequent phosphorylation by casein kinase II (dashed green arrow) but inhibits its subsequent phosphorylations by Pho85, proteins A and C (dashed blunted red lines) (55).Pho85 phosphorylation of Pah1 stimulates its subsequent phosphorylation by Rim11 (dashed green arrow) (56) but inhibits its subsequent phosphorylation by casein kinase I (dashed blunted red line) (55).The phosphorylations of Nem1-Spo7 by protein kinase A inhibits phosphorylation of Spo7 by protein kinase C, whereas the phosphorylation of the complex by protein kinase C inhibits phosphorylation of Nem1 by protein kinase A (dashed blunted red lines) (65,66).

Characterization of Hsl1 activity on Pah1
The kinase activity of Hsl1 was characterized by Pah1 as a substrate.The extent of Pah1 phosphorylation was dependent on the amount of Hsl1 and time of reaction (Fig. 5A and B, respectively), indicating that the protein kinase activity follows zero-order kinetics.In addition, the Hsl1 activity was dependent on the concentrations of Pah1 and ATP (Fig. 6A and B S1). B, cells expressing Hsl1 were grown to the indicated cell densities (A 600 ) in the SC medium.Lysates equivalent to 0.2 A 600 units of cells were used for immunoblot analysis with anti-calmodulin binding peptide antibody.The amount of Hsl1-TAP was quantified with the ImageQuant software.The data shown is the average of two independent experiments with individual data points shown.S1).Shown are only the phosphorylation sites that are confidently assigned at ≥ 1% of the total phosphopeptide abundances.
respectively).The V max and K m values of Hsl1 protein kinase with respect to Pah1 were 1.2 nmol/min/mg and 0.4 mM, respectively, and those for ATP were 2.5 nmol/min/mg and 31 mM, respectively.Overall, these enzymological properties support the conclusion that Pah1 is a bona fide substrate of the Hsl1 protein kinase.

Hsl1 phosphorylation of Pah1 inhibits its PAP activity
The phosphorylation of Pah1 by Hsl1 was examined for its effect on PAP activity.Immediately after its phosphorylation, Pah1 was measured for PAP activity by following the production of water-soluble 32 P i from chloroform-soluble 32 P ilabeled PA in the Triton X-100/PA-mixed micelles (30,90).Under this assay condition, PAP activity is dependent on the surface concentration of PA, but independent of its molar concentration (90).The phosphorylation of Pah1 caused a decrease in the catalytic efficiency of PAP activity; the V max of the phosphorylated enzyme (1.4 mmol/min/mg) was 5-fold lower than the unphosphorylated enzyme (7.2 mmol/min/mg) (Fig. 7).The K m value and Hill number for PA of the phosphorylated (3.5 mol% and n = 1.6, respectively) and unphosphorylated (2.5 mol% and n = 1.8, respectively) forms of Pah1 were not significantly different.

Hsl1 contributes to the Nem1-Spo7-mediated regulation of Pah1
The cellular effects of the Hsl1-mediated phosphorylation of Pah1 were examined by the analysis of cells expressing

Pah1 expression
The levels of the WT Pah1 and its phosphorylation-deficient forms were examined from cells in the exponential phase (Fig. 8).This growth phase is when Hsl1 is expressed (Fig. 3) and is expected to act on Pah1.Immunoblot analysis of the cell lysates with anti-Pah1 antibody confirmed that the phosphorylation-deficient variants are expressed in both pah1D and pah1D nem1D genetic backgrounds (Fig. 8, A and  B).In addition, their protein levels were not significantly different form that of WT Pah1 (Fig. 3).Compared with WT Pah1, the S748A variant showed a faster electrophoretic mobility, indicating that the serine phosphorylation affects Pah1 migration in SDS-PAGE (57).The S773A mutant, however, did not show a difference in the electrophoretic mobility.

Lipid synthesis
The pah1D and pah1D nem1D cells expressing the phosphorylation-deficient Pah1 were examined for lipid synthesis in the exponential phase by incubation with [2-14 C]acetate for 20 min.The incorporation of the radiolabel into each lipid species in the pulse labeling reflects a rate of synthesis (91).As described previously (33), the pah1D mutant showed a very low level of TAG synthesis (3%) and a resultant high level of phospholipid synthesis (68%) (Fig. 9).The expression of WT Pah1 complemented the pah1D mutant by increasing its TAG synthesis to the level of 20% but decreasing phospholipid synthesis to 37% (Fig. 9A).Similar to the effect of WT Pah1, the phosphorylation-deficient forms S748A and S773A complemented the defect of the pah1D mutant in lipid synthesis (Fig. 9A).In contrast to the expression of WT Pah1 in pah1D cells, its expression in pah1D nem1D cells showed a very weak complementation effect on TAG synthesis (Fig. 9B), which emphasizes the requirement of the Nem1-Spo7-mediated dephosphorylation of Pah1 to activate its function in lipid synthesis (57,92).Compared with WT Pah1, its phosphorylation-deficient forms S748A and S748A/S773A expressed in pah1D nem1D cells resulted in a significantly higher rate of TAG synthesis (Fig. 9B).For example, the mutant cells expressing the S748A/ S773A form exhibited a 117% increase in TAG synthesis and a 33% decrease in phospholipids synthesis (Fig. 9B).Interestingly, the S773A mutant alone did not show a significant effect on lipid synthesis.These results indicate that Pah1 deficient in phosphorylation on Ser-748 and Ser-773 partially bypasses the requirement of Nem1-Spo7 for its role in lipid synthesis.

Nuclear morphology
The mutants defective in Pah1 and/or Nem1-Spo7 display irregularly enlarged nuclei because of the increase in phospholipid synthesis and the aberrant expansion of the nuclear/ ER membrane (30,34,35,41,60,67) (Fig. 10A and B, vector control).The complementation of this phenotype was scored by fluorescence microscopy of the ER membrane marker Sec63-GFP (Fig. 10).Almost 90% of pah1D cells expressing WT or the phosphorylation-deficient mutant forms of Pah1 had a round nucleus (Fig. 10A).As described previously for the Nem1-Spo7 complex-deficient cells (60,67,93), only 15% of pah1D nem1D cells expressing WT Pah1 exhibited a round nucleus (Fig. 10B).In contrast, the phosphorylation-deficient forms expressed in pah1D nem1D cells had significant effects on the formation of a round nucleus (Fig. 10B).Compared with the number of cells expressing WT Pah1 (15%), the numbers of cells expressing the S748A, S773A, and S748A/ S773A mutants increased 44, 37, and 53%, respectively.substrate of Hsl1, a protein kinase associated with bud formation (70)(71)(72), and that phosphorylation of Pah1 by Hsl1 promoted phospholipid synthesis in the exponential phase of growth.Using Pah1 as substrate, Hsl1 activity followed zero order kinetics; the enzyme reaction was dependent on the amount of Hsl1 and time.The K m values of Hsl1 for Pah1 and ATP were in the low mmolar range and similar to those of other protein kinases acting on the protein substrate (Table 1).Ser-748 and Ser-773 were identified as the major sites of Pah1 phosphorylation with the latter site accounting for 76% of the phosphorylation.The serine phosphorylations together caused a 5-fold reduction in PAP activity, which is similar to the inhibitory effect of Pah1 phosphorylation by Pho85, a cyclindependent protein kinase that phosphorylates Ser-748 and 6 additional sites (50) (Fig. 2, Table 1).In contrast to Hsl1 and Pho85, the casein kinase I phosphorylation of Pah1 on Ser-748 stimulates its PAP activity (55).This stimulatory effect might be explained by Pah1 phosphorylation on the non-overlapping sites that regulate PAP activity in a manner opposite to that of Hsl1 (55) (Fig. 2, Table 1).Phosphorylation of Pah1 by protein kinases A ( 52) and C ( 53), both of which share Ser-773 as a phosphorylation site with Hsl1, causes a moderate inhibition of PAP activity, and the contribution of Ser-773 to the overall inhibition by either protein kinase is very small (52,53).Accordingly, we speculate that the inhibitory effect of Hsl1 on Pah1 activity is mainly due to its phosphorylation on Ser-748, which accounts for only 24% of the total phosphorylation.The variation between the effects of phosphorylation by the protein kinases, despite acting on the common target sites, highlights the complex nature of the phosphorylation-mediated regulation of Pah1 function.That multiple protein kinases target the same residue emphasizes the importance of maintaining its phosphorylation when they are active at different times and growth conditions.

Discussion
In the pulse labeling of lipid synthesis, the lack of Ser-748 phosphorylation alone, and in combination with the loss of Ser-773 phosphorylation, caused a significant increase in TAG synthesis with a corresponding decrease in phospholipid synthesis.These mutational effects, which were notable in the nem1D mutant, indicate the requirement of Pah1 phosphorylation at specific sites for its regulation by the Nem1-Spo7 complex (50,51,57,61).The S773A mutation was not as effective as the S748A mutation in the absence of the Nem1-Spo7 complex, but the combined mutations had a stronger effect on Pah1 function (e.g., TAG synthesis and nuclear morphology).The mutational effects of S748A and S773A, which bypass the requirement of Nem1-Spo7 for Pah1 function (57,60,61), are similar to those shown by Pah1-7A (50,51), which contains non-phosphorylable alanine residues in place of seven residues including Ser-748 phosphorylated by Pho85 (50).
Although Pah1 mutants that lack the target sites of Hsl1 or Pho85 exhibit a gain-of-function phenotype in the absence of Nem1-Spo7, they show a difference with respect to the role of phosphorylation in protein stability/degradation.In general, phosphorylated Pah1 is protected against its degradation by the 20S proteasome, whereas the enzyme dephosphorylated by Nem1-Spo7 is susceptible to proteasomal degradation (59).The phosphorylation sites of Pah1 that are most affected by proteasomal degradation are the seven sites targeted by Pho85 (50,59).For example, when Pah1-7A is expressed in pah1D and pah1D nem1D cells, its abundance is greatly reduced when compared with that of the WT protein (50).In striking contrast, the protein levels of the phosphorylation-deficient S748A and S773A mutants, in both genetic backgrounds, were similar to that of the WT control.This indicates that the sites of Pah1 phosphorylated by Hsl1 do not play a key role in controlling the protein stability.
Hsl1 is also known to engage in genetic interactions with other members of the Pah1-regulating network of protein kinases.Hsl1 exhibits a genetic interaction with Slt2, a MAP kinase that is activated by protein kinase C in a signaling cascade that is active when the morphogenesis checkpoint is triggered (94).Protein kinase C activity is dependent on DAG produced by Pah1 (95), highlighting the importance of Pah1 during exponential growth.Hsl1 also has a functional relationship with Cdc28.A major function of Hsl1 is to downregulate Swe1, a Cdc28 inhibitor, suggesting that Hsl1 and Cdc28 are active at the same time and therefore may phosphorylate Pah1 at the same time (96).Swe1 is also predicted to phosphorylate Pah1 (69), raising a possibility that its phosphorylation by Swe1 has an opposing effect on Pah1 when compared to its phosphorylation by Hsl1 and Cdc28.The condition in which Swe1-dependent phosphorylation of Pah1 stimulates its PAP activity would prevent the biogenesis of membrane phospholipids while Cdc28 is inhibited and cell growth is arrested.
The in vivo phosphorylation of Pah1 by Hsl1 is also likely to be affected by other protein kinases that phosphorylate Hsl1.Hsl1 phosphorylates PA phosphatase Pah1 Like Pah1, Hsl1 is subject to regulation by multiple phosphorylation (97).The premise that the protein kinases acting on Pah1 may also phosphorylate each other was previously raised for Rim11, which appears to be phosphorylated by Pho85 in reactions where both protein kinases are present along with the substrate Pah1 (56).Protein kinases known to phosphorylate Hsl1 include Cdc28, a protein kinase for which Pah1 phosphorylation has been characterized (51), and Mck1, a glycogen synthase kinase 3b homolog that is predicted to phosphorylate Pah1 (69,79,98).
In addition to the Pah1-targeting protein kinases, Hsl1 is phosphorylated by the protein kinase Elm1; phosphorylation at the activation loop Thr-273 is required for Hsl1 protein kinase activity (87).Interestingly, the activation loop is found in at least two other protein kinases, the AMP-activated kinase Snf1 and the spindle position checkpoint protein kinase Kin4, both of which are predicted to phosphorylate Pah1 (69,99,100).This raises a possibility that Elm1 has a larger role in lipid metabolism that is mediated by its activation of Pah1-targeting protein kinases, which would be consistent with a lipidomics study showing that glycerophospholipid levels are altered in the absence of Elm1 (101).Similar to Hsl1, both Snf1 and Kin4 have functions related to cellular proliferation during the exponential phase of growth.Further studies into Pah1 phosphorylation by Kin4 and Snf1 will elucidate the mechanism of Elm1-dependent regulation of lipid metabolism and may give insight into whether these Elm1-regulated protein kinases have a synergistic effect on Pah1 phosphorylation during cell proliferation.
The mammalian homolog of S. cerevisiae Pah1 is known as lipin (30).The three spliced variants of lipin 1 (a, b, and g), along with the lipin 2 and 3 isoforms are PAP enzymes (30,102,103).The critical roles that PAP activity plays in humans and mice are typified by assorted abnormalities (e.g., lipodystrophy, insulin resistance, peripheral neuropathy, rhabdomyolysis) that result from the loss of the lipin 1 enzyme (104-109).In the mouse model, excess lipin 1 results in the obese phenotype (104,110).Like Pah1, lipins 1 and 2 are subject to multiple phosphorylation (111)(112)(113)(114)(115), and for at least lipin 1, the enzyme is dephosphorylated by the mammalian counter part of the Nem1-Spo7 protein phosphatase complex known as CTDNEP1-NEP1-R1 (108,(116)(117)(118)(119). As in S. cerevisiae, the phosphorylation state of lipin 1 governs its subcellular localization (111-113, 120, 121).Mammalian homologs of Hsl1 are BRSK1 and 2. These serine/threonine protein kinases are specifically expressed in the brain and play key roles in neuronal development (122,123).Whether they phosphorylate and regulate lipin activity and/or its localization is unclear, but the conservation of the S. cerevisiae Nem1-Spo7/Pah1 phosphatase cascade in mammalian cells suggests an important avenue of investigation.

Experimental procedures Reagents
Avanti Polar Lipids and Analtech, respectively, supplied lipids and silica gel GHL TLC plates.DNA size ladders, molecular mass protein standards, and reagents for electrophoresis and Western blotting were from Bio-Rad.BioSynthesis, Inc prepared the rabbit anti-Pah1 antibody directed against the sequence TSIDKEFKKLSVSKAGA (residues 778-794) (51).Cayman Chemical was the supplier of protease inhibitors leupeptin and pepstatin.Carrier DNA for yeast transformations was from Clontech.Growth media were purchased from Difco Laboratories.InstantBlue Coomassie stain was from Expedeon.GE Healthcare was the supplier of Q-Sepharose, IgG-Sepharose, polyvinylidene difluoride membrane, and the chemifluorescence Western blotting detection kit.Mouse anti-Pgk1 antibody (product number: 459250; lot number: E1161) was from Invitrogen.MilliporeSigma supplied ATP, bovine serum albumin, Ponceau S stain, protease and phosphatase inhibitors, Triton X-100, rabbit anti-calmodulin binding protein epitope tag antibody (product no.07-482, lot no.3467112), and alkaline phosphatase-conjugated goat antimouse IgG antibody (product no.A3562; lot no.SLBG1482V).National Diagnostics was the supplier of scintillation counting supplies.Q5 site-directed mutagenesis kit and other reagents for DNA manipulations were from New England Biolabs.Radiochemicals were purchased from Perki-nElmer Life Sciences.DNA gel extraction and plasmid purification kits and the nickel-nitrilotriacetic acid agarose resin were from Qiagen.Thermo Fisher Scientific was the source of Pierce mass spectrometry grade proteases and strong anion exchange spin columns, alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (product no.31340, lot number: NJ178812), and S. cerevisiae strain BY4741-HSL1-TAP.All other chemicals were reagent grade.

Strains, plasmids, and growth conditions
The strains and plasmids used in this study are listed in Table 2. S. cerevisiae strain BY4741-HSL1-TAP was used for the purification of TAP-tagged Hsl1.Strains SS1026 and SS1132 were used for the expression of the WT and phosphorylation-deficient mutant forms of Pah1 in the presence and absence of the Nem1-Spo7 protein phosphatase complex, respectively.E. coli strains DH5a and BL21(DE3) pLysS were used for the propagation of plasmids and heterologous expression of His 6 -tagged Pah1, respectively.Plasmids containing PAH1 (S748A, S773A, and S748A/S773A) were generated from pGH315 using the Q5 Site-directed mutagenesis kit.Standard methods were used for the isolation of plasmid DNA, for digestion and ligation of DNA, and for PCR amplification of DNA (124)(125)(126).Plasmid transformations of E. coli (125) and S. cerevisiae (127) were performed by standard methods.DNA constructs were confirmed by PCR analysis and DNA sequencing.
S. cerevisiae cells were cultured using standard methods (124,125).Solid media plates contained 2 or 1.5% agar for the growth of S. cerevisiae or E. coli, respectively.Cells expressing TAP-tagged Hsl1 were grown at 30 C in 2 L YEPD (1% yeast extract, 2% peptone, 2% glucose) medium.Growth in liquid medium was monitored by absorbance at 600 nm (A 600 ) using a spectrophotometer.S. cerevisiae cells carrying a plasmid were grown at 30 C in a synthetic drop-out medium, which lacks a specific amino acid from a synthetic complete (SC) medium for the plasmid selection.The E. coli cells were grown at 37 C in lysogeny broth (LB) medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0); ampicillin (100 mg/ml) was added to select for cells carrying plasmids.For His 6 -tagged Pah1 expression, the bacterial cells harboring plasmid pGH313 were grown to A 600 0.5 at room temperature in 500 ml of LB medium containing ampicillin (100 mg/ml) and chloramphenicol (34 mg/ml); expression was induced for 1 h with 1 mM isopropyl-b-D-thiogalactoside (30).

Preparation of cell lysates and enzyme isolations
Yeast cells were collected by centrifugation at 1500g for 5 min.The cells were washed with water and boiled in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 10% glycerol, 10 mM 2-mercaptoethanol, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 mg/ml aprotinin, 5 mg/ml leupeptin, 5 mg/ml pepstatin, and 8% SDS.The cell lysates were directly used for SDS-PAGE and immunoblotting.All procedures for enzyme purification were conducted at 4 C. His 6 -tagged Pah1 expressed in E. coli was purified to near homogeneity from cell extracts by affinity chromatography with nickel-nitrilotriacetic acid-agarose (30), followed by ion exchange chromatography with Q-Sepharose (61).TAP-tagged Hsl1 was partially purified by affinity chromatography with IgG-Sepharose (73,128).S. cerevisiae cells expressing TAP-tagged Hsl1 were harvested, and the cell pellet was resuspended in 50 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 1 mM EDTA, and Roche EDTA-free protease inhibitors.The cells were then lysed with glass beads using a Mini-Beadbeater-16 (5 repeats of 1-min burst with 2-min cooling between bursts).The cell lysate was centrifuged at 1500g for 10 min, and the supernatant was mixed with an equal volume of 50 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 1 mM EDTA, Roche EDTAfree protease inhibitors, and 2% Triton X-100 and centrifuged at 100,000g for 1 h.The supernatant was applied to a 0.5 ml IgG-Sepharose column equilibrated with 50 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 0.5 mM EDTA, and 0.1% Triton X-100.The column was washed with the equilibration buffer with 10 mM Tris-HCl (pH 8.0) to lower buffer capacity, and fusion protein was eluted with 50 mM glycine (pH 3.0) and 0.1% Triton X-100 (55) and neutralized by the addition of 0.2 volume of 1M Tris-HCl (pH 8.0).The final protein concentration of the purified enzyme was 1 mg/ml.The purified enzyme preparation was stored at −80 C after the addition of glycerol to a final concentration of 10%.

Protein kinase assay
Hsl1 protein kinase activity was measured at 30 C by following the incorporation of radioactive phosphate from [g-32 P]ATP into Pah1 in a total volume of 20 ml as described previously (51).The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 100 mM [g-32 P]ATP (3000 cpm/pmol), 0.25 mM Pah1, 2 mM dithiothreitol, and the indicated amount of Hsl1.The phosphorylation reaction was terminated by the addition of 5 ml 5x Laemmli sample buffer, followed by SDS-PAGE (129) to resolve the 32 P-labeled Pah1 from radioactive ATP.The phosphorylated Pah1 was visualized by phosphorimaging using a Storm 860 Molecular Imager (GE Healthcare) and the extent of phosphorylation was quantified by ImageQuant software.

Identification of Hsl1 peptide sequences and phosphorylation site analysis of Pah1 by LC-MS/MS
To confirm the identity of TAP-tagged Hsl1, the fusion protein contained within an SDS polyacrylamide gel slice was digested with trypsin at 37 C followed by the analysis of the digest by LC-MS/MS (89) (Table S1).The amino acid residues of Pah1 phosphorylated by the Hsl1 protein kinase were analyzed by LC-MS/MS.The proteolytic digestion of the Hsl1phosphorylated Pah1 in polyacrylamide gel slices, analysis of peptide fragments by LC-MS/MS, and database analysis were performed as described previously (89) (Table S1) at the Center for Integrative Proteomics Research at Rutgers Hsl1 phosphorylates PA phosphatase Pah1 University.The raw data and database results for the peptide analyses of Pah1 and Hsl1 are deposited in the MassIVE repository (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) with the accession number MSV000095027.

SDS-PAGE and Western blot analysis
Standard procedures were used for SDS-PAGE (129) and Western blotting (130,131).The samples for Western blotting were normalized to total protein loading.Protein transfer from polyacrylamide gels to PVDF membranes was monitored by staining with Ponceau S. Rabbit anti-calmodulin binding peptide, rabbit anti-Pah1, and mouse anti-Pgk1 antibodies were used at a final concentration of 2 mg/ml.A dilution of 1:5000 was used with the secondary goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody that is conjugated with alkaline phosphatase.The enhanced chemifluorescence immunoblotting substrate was used to detect immune complexes.Fluorimaging with a Storm 865 Molecular Imager was used to visualize fluorescence signals from immunoblots; image intensities were analyzed by ImageQuant TL software (GE Healthcare).A standard curve ensured that the immunoblot signals were in the linear range of detection.

PAP assay
PAP activity was measured by following the release of watersoluble 32 P i from chloroform-soluble [ 32 P]PA (10,000 cpm/ nmol) using the Triton X-100/PA mixed micellar assay as described by Carman and Lin (132).The standard reaction mixture contained 50 mM Tris-HCl (pH 7.5), 1 mM MgCl 2 , 0.2 mM PA, 2 mM Triton X-100, and enzyme protein in a total volume of 100 ml.The surface concentrations of PA (mol %) were obtained by maintaining the molar concentration of PA at 0.2 mM and varying the molar concentrations of Triton X-100 (132).Enzyme assays were performed in triplicate and all reactions were linear with time and protein concentration.[ 32 P]PA was enzymatically produced from DAG by DAG kinase with [g-32 P]ATP (132).

Protein determination
The protein-dye binding assay of Bradford (133) was used to estimate protein concentration; bovine serum albumin was used as the standard.
Lipid labeling and analysis S. cerevisiae cells were pulse-labeled with [2-14 C]acetate for 20 min (33,134).Lipids were extracted from cells (135,136) and resolved by one-dimensional TLC on silica gel plates using the solvent system hexane/diethyl either/glacial acetic acid (40:10:1, v/v) (137).The resolved lipids were visualized by phosphorimaging with a Storm 860 Molecular Imager (GE Healthcare) and quantified by ImageQuant software using a standard curve of [2-14 C]acetate.The position of radiolabeled lipids on the TLC plate was confirmed by comparison with the migration of authentic standards visualized by staining with iodine vapor.

Microscopic analysis of nuclear morphology
Nuclear/ER morphology was examined by fluorescence microscopy of cells expressing the SEC63-GFP plasmid (44).The percentage of cells with a round nucleus was scored from ≥4 fields of view (≥200 cells).The microscope used to image the cells was a Nikon Eclipse Ni-U microscope with the EGFP/ FITC/Cy2/AlexaFluor 488 filter and fields of view were recorded by a DS-Qi2 camera.Image analysis was performed with NIS-Elements BR software.

Data analysis
The statistical analysis of data was determined with Microsoft Excel software.The p values < 0.05 were taken as a significant difference.The enzyme kinetics module of Sigma-Plot software was used to analyze kinetic data.

Figure 1 .
Figure 1.Roles of Pah1 in lipid synthesis.The structures of CDP-DAG, PA, DAG, and TAG are shown with fatty acyl groups of 16 and 18 carbons with and without a single double bond where indicated.Pah1 plays a key role in the use of PA for the synthesis of membrane phospholipids via CDP-DAG or the synthesis of TAG via DAG.The PAP reaction is counterbalanced by the CTP-dependent conversion of DAG to PA by the diacylglycerol kinase Dgk1.The DAG produced by the PAP reaction may be used for the synthesis of phosphatidylcholine and phosphatidylethanolamine via the CDP-choline and CDPethanolamine branches, respectively, of the Kennedy pathway when cells are supplemented with choline and/or ethanolamine.In addition to its role in lipid synthesis, PA signals the transcriptional regulation of phospholipid synthesis genes via the Henry regulatory circuit.More comprehensive pathways of lipid synthesis, along with details of the Henry regulatory circuit may be found in Refs.(5,6).CL, cardiolipin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine.

Figure 2 .
Figure 2. Pah1 domains, regions, and phosphorylation sites; impacts and interrelationships of phosphorylation and dephosphorylation on Pah1 function.A, the schematic shows the domains/regions of Pah1.The N-LIP and the haloacid dehalogenase (HAD)-like domains are required for PAP activity(30,41).The N-terminal amphipathic helix (AH) is responsible for interaction with the membrane(48).The regulation of the phosphorylation (RP) domain facilitates phosphorylation(93).The essential tryptophan contained within the WRDPLVDID domain, which is C-terminal to the HAD-like domain(138), is required for the in vivo catalytic function(89,138).The acidic tail (AT) associates with the Nem1-Spo7 complex (62) through ionic interaction with the Cterminal basic tail of Spo7(139).The intrinsically disordered regions (IDRs) contain almost all of the sites of phosphorylation that serve for interaction with the Nem1-Spo7 complex.The serine (S) and threonine (T) residues known to be phosphorylated(50-57, 75, 83, 84, 89, 140-147) are grouped at their approximate regions in the Pah1 protein.The sites phosphorylated by casein kinase I (CKI)(55), casein kinase II (CKII) (54), Cdc28 (51), Hsl1 (this study), Pho85 (50), protein kinase A (PKA)(52), protein kinase C (PKC)(53), and Rim11 (56) are indicated.B, the positive (solid green arrow) or negative (solid blunted red line) impacts of Pah1 phosphorylation (denoted by the small black circles) by the indicated protein kinases and Pah1 dephosphorylation by Nem1-Spo7, whose components are also subject to phosphorylation by protein kinases A and C (65, 66), are indicated.The casein kinase I phosphorylation of Pah1 stimulates its subsequent phosphorylation by casein kinase II (dashed green arrow) but inhibits its subsequent phosphorylations by Pho85, proteins A and C (dashed blunted red lines)(55).Pho85 phosphorylation of Pah1 stimulates its subsequent phosphorylation by Rim11 (dashed green arrow) (56) but inhibits its subsequent phosphorylation by casein kinase I (dashed blunted red line)(55).The phosphorylations of Nem1-Spo7 by protein kinase A inhibits phosphorylation of Spo7 by protein kinase C, whereas the phosphorylation of the complex by protein kinase C inhibits phosphorylation of Nem1 by protein kinase A (dashed blunted red lines)(65,66).

Figure 3 .
Figure 3. Isolation of Hsl1 and growth-dependent expression of the protein.A, the isolated Hsl1 was subjected to SDS-PAGE (10% polyacrylamide gel) and stained with Coomassie blue (left).The enzyme preparation was also subjected to immunoblot analysis using calmodulin-binding peptide antibody (right).The positions of molecular mass standards and Hsl1 are indicated.The Hsl1 protein from a replicate gel was used for proteolytic digestion and phosphopeptide identification by LC-MS/MS analysis (TableS1).B, cells expressing Hsl1 were grown to the indicated cell densities (A 600 ) in the SC medium.Lysates equivalent to 0.2 A 600 units of cells were used for immunoblot analysis with anti-calmodulin binding peptide antibody.The amount of Hsl1-TAP was quantified with the ImageQuant software.The data shown is the average of two independent experiments with individual data points shown.

Figure 4 .
Figure 4. Hsl1 phosphorylates Pah1 on Ser-748 and Ser-773.A, 1 mg Pah1 was incubated for 10 min at 30 C with 100 mM [g-32 P]ATP (3000 cpm/pmol) in the absence (−) and presence (+) of 5 ng Hsl1.The reaction mixtures were resolved by SDS-PAGE (10% polyacrylamide gel) and subjected to protein staining with Coomassie blue (lower), followed by phosphorimaging (upper).The data shown are representative of three experiments.B, Pah1 phosphorylated by Hsl1 was extracted from an SDS-polyacrylamide gel, reduced, and alkylated, followed by digestion with trypsin; the resulting peptides were analyzed by LC-MS/MS.The abundance of phosphopeptides containing the indicated phosphorylation sites was estimated from intensities reported by Proteome Discoverer and expressed as a percentage of the intensities of all phosphopeptides identified for the protein (TableS1).Shown are only the phosphorylation sites that are confidently assigned at ≥ 1% of the total phosphopeptide abundances.

Figure 5 .Figure 6 .
Figure 5. Hsl1 protein kinase activity on Pah1 is dependent on the amount of Hsl1 and the reaction time.Pah1 was incubated at 30 C with Hsl1 and [g-32 P]ATP, subjected to SDS-PAGE, and analyzed by phosphorimaging.The enzyme assay was conducted by varying the amount of Hsl1 (A) and the reaction time (B).A, 500 nM Pah1/100 mM ATP/10 min; B, 500 nM Pah1/100 mM ATP/5 ng Hsl1.The data shown are means ± S.D. (error bars) from triplicate assays.

Figure 7 .Figure 8 .
Figure 7. Hsl 1 phosphorylation of Pah1 inhibits its PAP activity. 1 mg Pah1 was phosphorylated by 5 ng Hsl1 for 2 h with 100 mM ATP in a total volume of 20 ml.The unphosphorylated Pah1 as control was incubated under the same reaction condition in the absence of Hsl1.After the incubation, 10% of the reaction mixture was measured for PAP activity.The surface concentration of PA (mol %) was adjusted by maintaining the molar concentration of PA at 0.2 mM and varying the molar concentration of Triton X-100 (90).The data shown are means ± S.D. (error bars) from triplicate assays.p-Pah1, phosphorylated Pah1.

Figure 9 .Figure 10 .
Figure 9. Hsl1 phosphorylation-deficient S748A and S773A mutations in Pah1 cause a decrease in the synthesis of phospholipids and an increase in TAG synthesis in pah1D nem1D mutant cells.The pah1D (A) and pah1D nem1D (B) cells expressing the indicated WT and Hsl1 phosphorylation-deficient S748A, S773A, and S748A/S773A mutant forms of Pah1 were grown at 30 C to the exponential phase in 2 ml of SC-Leu medium.The cells were then incubated with [2-14 C]acetate (1 mCi/ml) for 20 min.Lipids were extracted, separated by one-dimensional TLC, and subjected to phosphorimaging, followed by ImageQuant analysis.The percentages shown for phospholipids and TAG were normalized to the total 14 C-labeled chloroform-soluble fraction.The data are means ± S.D. (error bars) from three separate experiments.The individual data points are also shown.*, p < 0.05 versus WT.

Table 1
Kinetic properties of protein kinases that phosphorylate Pah1

Table 2
Strains and plasmids used in this study